GSM6021983: P13_GFP+ve; Mus musculus; RNA-Seq
Fresh brain tissues from embryo/postnatal Sox2eGFP mice were collected following intracardial perfusion of mice (pregnant females in case of embryo samples) with PBS. The tissue was transferred to a petri dish placed on ice and the cerebrum was isolated under a Leica stereoscope, rinsed in PBS, dissociated into single cells as previously reported then passed through a 40-µm mesh cell strainer. Debris was removed using a debris-removal kit (Miltenyi) and the cells were stained with DAPI followed by sorting the live GFP+ve/-ve cells. Single-cell suspensions with an average cell viability of 80% were loaded onto the 10x Genomics Chromium Platform (10X Genomics, Pleasanton, CA, USA) to recover 6,000 single-cell-containing gel beads in emulsion (GEMs). Single Cell RNA-seq libraries were prepared according to 10x Genomics manufacturing protocols (Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075. User Guide CG000183 Rev A) using a Veriti thermal cycler (ThermoFisher Scientific, Waltham, MA, USA). cDNA and Library quality were assessed using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Quantification of Library was performed using StepOne Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Sequencing was done on the Illumina Hiseq 2500 paired-end sequencing 29+8+101bp.